PROJECT SUMMARY/ABSTRACT Clostridium difficile is the main cause of antibiotic-associated diarrhea. Since 2003, the incidence and severity of C. difficile infection (CDI) has risen in the U.S. and globally. In the U.S. in 2011, there were ~29,000 deaths from ~500,000 CDI cases. In 2013 the CDC designated C. difficile as an Urgent Threat. These trends were related to the emergence of epidemic strains, in particular epidemic 027. Unfortunately, 20% or more of patients treated with the antibiotics metronidazole or vancomycin, undergoes a recurrence, which is a further frustrating feature of managing CDI, as the prognosis for patients with recurrent CDI is often poor. CDI recurrence partly results from damage of the intestinal microbiota community by metronidazole or vancomycin. There is a need for alternative treatments that are specific to disarming C. difficile without harming the gut microbiota. This is addressed in the proposed study, which seeks to develop a novel high-throughput screening (HTS) platform for discovering non-antibiotic compounds that specially inhibits C. difficile virulence, namely inhibiting cellular production of toxins that are responsible for tissue damage and spores that are responsible for recurrence. Preliminary data shows that such molecules do not harm the microbiota and reduce the onset of recurrent CDI in animals. The study proposes to establish a HTS platform for toxin synthesis inhibitors, which is feasible owing to state-of-the art facilities and resources at institutions involved in the study i.e. Texas A&M Health Science Center, St Jude Children?s Research Hospital, Baylor College of Medicine and supporting research cores in the Texas Medical Center. The team deploys a large compound collection for HTS, automated and semi-automated screening technologies, and novel in vitro and in vivo microbiota models that are clinically reflective of CDI. The studies outcome will be one or more candidate scaffolds that are suitable for lead optimization and drug development. These molecules will help to probe ill-defined molecular pathogenic mechanisms to understand the biology of this organism. Public health. The successful completion of this study impacts healthcare by advancing candidate molecules for drug development to save lives by reducing the onset of CDI.